Milk is well known as a balanced nutrient, and one of the best sources of calcium. Calcium is the most abundant metal in the mammalian body, is absorbed by humans and other mammals in the formation of bones and teeth, and is involved in various metabolic reactions in the body. The beneficial effects of milk are due not only to the calcium content, but also to the high efficacy or utilization of the calcium in terms of the ability of milk calcium to be digested and absorbed. These characteristics are attributable to lactose, calcium-binding protein, and casein.
Calcium absorption occurs both by active transport in the upper part of the small intestine, regulated by vitamin D and hormones, and by passive transport in the lower part of the small intestine. Naito, H. A., Chemistry and Biology 18,554-558 (1980). It is widely accepted that calcium should be solubilized to be absorbed by the small intestine.
It has been suggested that a phosphopeptide produced when casein is digested by pancreatic enzymes, particularly trypsin, solublizes calcium in the small intestine, thus increasing the amount of soluble calcium and working to enhance calcium absorption. Naito, H. A. et al., Agricultural and Biolog. Chem. 36, 409-415 (1972); and Lee, Y. S. et al., British J. Nutrition 43, 457-467 (1980). The peptide is referred to as casein phosphopeptide or CPP and may be derived from .beta.- or .alpha.- casein. The structures and some characteristics of the CPPs are shown in Table I. .beta.-CPP has been studied by Manson, A. et al., Archives of Biochem. and Biophysics 145, 16-26 (1971). The molecular weight and some data for .alpha.-CPP is estimated from the reference. It is apparent that CPP preparations can be heterogeneous, and CPP may be further digested as compared to the data in the table.
TABLE 1 ______________________________________ .alpha.-cpp .beta.-cpp ______________________________________ Molecular about 4,552 about 3,123 Weight Structure H.sub.2 N.AsP--Ile--Gly-- H.sub.2 N.Arg--Glu--Leu-- Ser.sup.p --Glu--Ser.sup.p --Thr-- Glu--Glu--Leu--Asn-- Glu--Asp--Gln--Ala-- Val--Pro--Gly--Glu-- Met--Glu--Asp--Ile-- Ile--Val--Glu-- Lys--Gln--Met--Glu-- Ser.sup.p --Leu--Ser.sup.p -- Ala--Glu--Ser.sup.p --Ile-- Glu--Glu--Ser--Ile-- Ser--Ser.sup.p --Ser.sup.p --Glu-- Thr-- Glu--Ile--Val--Pro-- Arg.COOH Asn--Ser.sup.p --Val--Glu-- Gln--Lys.COOH Number of 6 4 phosphoric acid residues N/P 7.2 8 atomic ratio Number of 12 7 Carboxyl residues Isoelectric pH 1-2 pH 1-2 point ______________________________________
In a prior art method, casein is hydrolyzed with a crystalline trypsin to produce CPP, after which the reaction mixture is adjusted to a pH value of 4.5-5.0 to eliminate the unreacted casein and part of the impurities by 5 precipitation, followed by addition to the supernatant of barium chloride and ethanol (to the final concentration of 50 v/v %) to precipitate and recover CPP. R. F. Peterson, L. W. Nouman and T. L. McMeekin, Journal of the American Chemical Society, 80, 95-99 (1958).
The CPP of Peterson is not acceptable for a beverage because barium chloride is not an approved food additive, in contrast to calcium chloride. CRC Handbook of Food Additives (2d Ed. 1972), pp. 798-799, 808-809. To similar effect is Mellander, "On chemical and nutritional differences between casein from human and from cow's milk," Upsala Lakareforenings Forhandlingar pp. 182-195 (1947). There, preparations of CPP, including calcium salts, are prepared with toxic substances such as lead acetate that are not acceptable in food products.
Alternatively, CPP produced by hydrolysis of casein with a crystalline trypsin may be fractionated and separated by ultrafiltration or chromatographic techniques such as gel permeation chromatography or ion-exchange chromatography. Brule et al., U.S. Pat. No. 4,740,462. The Brule method uses a plurality of enzymes and produces a preparation with smaller CPP peptides with molecular weights predicted to be generally below 2500 daltons. The ultrafiltration and chromatography methods of Brule and others may have some research utility, but are not as suitable or economical on an industrial scale as precipitation methods such as according to the invention.
The characteristics of the CPP preparation produced by each method differ, in terms of e.g. molecular weight, nitrogen/phosphorous ratio, and purity.
Prior art preparations of CPP have had some use in solid foods and opaque beverages such as milk, natural fruit juice, canned juice and canned coffee. However, it has not been possible to prepare a storage stable transparent acid drink containing a pure preparation of CPP.
The following requirements are called for in a transparent acid drink. First, when raw materials used in the acid drink are mixed, dissolved, heat-sterilized and cooled, the resulting mixed solution must be completely transparent. Next, the transparent appearance of the mixed solution must be maintained even during storage. Generally, when prior art CPP is added to an acidic drink (pH 2.0-4.0) followed by mixing, dissolving, pH adjustment, heat sterilization and cooling, a precipitate or sediment forms, causing the drink to become turbid and to lose its transparency.